Method for measurement using sodium azide

ABSTRACT

A method of measuring an analyte in a sample with excellent sensitivity using a redox reaction is provided. In this method, a reducing substance or an oxidizing substance derived from the analyte is measured in the presence of a tetrazolium compound and sodium azide using the redox reaction, and an amount of the analyte is determined from the amount of the reducing substance or oxidizing substance thus measured. The tetrazolium compound and the sodium azide are present at a ratio in a range from 20:3 to 20:12. Preferably, a solution containing the tetrazolium compound and the sodium azide is aged and then added to the sample.

TECHNICAL FIELD

[0001] The present invention relates to a method for measurement using aredox reaction.

BACKGROUND ART

[0002] Conventionally, measurement of the amount of an analyte in asample using a redox reaction has been utilized for a wide range ofapplications. For example, such measurement has been utilized formeasuring glycated proteins in applications such as biochemicalanalyses, clinical tests, and the like.

[0003] For example, glycated proteins in blood, especially glycatedhemoglobins in erythrocytes, serve as important indexes in thediagnosis, treatment, etc. of diabetes, because they reflect thepatient's past history of blood glucose levels. Such glycated proteinsin erythrocytes are measured utilizing a redox reaction, for example, inthe following manner.

[0004] First, erythrocytes are hemolyzed to prepare a sample. Then, thishemolyzed sample is treated with a fructosyl amino acid oxidase(hereinafter referred to as “FAOD”) so that the FAOD acts on a glycationsite of a glycated protein to form hydrogen peroxide. The amount of thehydrogen peroxide corresponds to the amount of the glycated protein.Subsequently, a peroxidase (hereinafter referred to as “POD”) and areducing agent are added to the sample, so that a redox reaction occursbetween the hydrogen peroxide and the reducing agent with the POD as acatalyst. At this time, when a reducing agent that develops color whenit is oxidized is used, the amount of the hydrogen peroxide can bedetermined by measuring the color developed. As a result, the amount ofthe glycated protein in the erythrocytes can be determined.

DISCLOSURE OF INVENTION

[0005] However, depending on the sample used, the conventional methodsmay not exhibit sufficient measurement sensitivity and thus may fail toimprove the accuracy of the measurement. Furthermore, since glycatedproteins in blood serve as important indexes in the diagnosis,treatment, etc. of diabetes as described above, still furtherimprovement in the accuracy of measurement is desired in methods ofmeasuring them using a redox reaction.

[0006] Therefore, it is an object of the present invention to provide amethod of measuring an analyte in a sample with high sensitivity using aredox reaction.

[0007] In order to achieve the above object, the present inventionprovides a method of measuring an analyte in a sample using a redoxreaction, including: measuring an amount of a reducing substance or anoxidizing substance derived from the analyte in the presence of atetrazolium compound and sodium azide using the redox reaction; anddetermining an amount of the analyte from the amount of the reducingsubstance or oxidizing substance thus measured. By carrying out themeasurement in the presence of the tetrazolium compound and the sodiumazide as described above, the measurement sensitivity can be improved,although the mechanism is unknown. In the present invention, “a reducingsubstance or an oxidizing substance derived from an analyte” includesthe analyte itself, a reducing/oxidizing substance contained therein,and a reducing/oxidizing substance formed from the analyte using anoxidoreductase or the like.

[0008] In the method of the present invention, it is preferable that thetetrazolium compound (A) and the sodium azide (B) are present at a ratio(molar ratio A:B) in a range from 20:3 to 20:12.

[0009] In the method of the present invention, it is preferable that afinal concentration of the tetrazolium compound in a reaction solutionof the redox reaction is in a range from 0.5 to 2.5 mmol/l, and a finalconcentration of the sodium azide in the reaction solution is in a rangefrom 0.13 to 1.3 mmol/l.

[0010] In the method of the present invention, it is preferable that asolution containing the tetrazolium compound and the sodium azide isaged and is then added to the sample because this allows still furtherimprovement in the sensitivity.

[0011] It is preferable that the solution is aged at a temperature in arange from 20° C. to 60° C. Furthermore, it is preferable that thesolution is aged for at least 6 hours, more preferably for 6 to 120hours.

[0012] In the method of the present invention, it is preferable that thetetrazolium compound is2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt.

[0013] In the method of the present invention, it is preferable that theoxidizing substance derived from the analyte is hydrogen peroxide, andthat the amount of the hydrogen peroxide is measured by the redoxreaction. The amount of the hydrogen peroxide preferably is measuredusing an oxidase and a substrate that develops color by oxidation(hereinafter, referred to as a color-developing substrate), for example.

[0014] In the method of the present invention, the type of the sample isnot particularly limited. The method also can be applied to samplesother than whole blood, plasma, serum, and blood cells, e.g., biologicalsamples such as urine and spinal fluid, drinks such as juices, foodssuch as soy sauce and Worcestershire sauce.

[0015] Furthermore, the analyte is not particularly limited as long as aredox reaction is utilized. For example, the analyte may be componentsin whole blood, components in erythrocytes, components in plasma,components in serum, components in urine, components in spinal fluid,and the like, and it is preferably a component in erythrocytes. Forexample, when a component in erythrocytes is to be measured, whole blooditself may be hemolyzed to prepare a sample, or erythrocytes may beseparated from whole blood and hemolyzed to prepare a sample. Examplesof the analyte include glycated proteins such as glycated hemoglobinsand glycated albumins, glycated peptides, glycated amino acids, glucose,uric acid, cholesterol, creatinine, sarcosine, and glycerol. Amongthese, glycated proteins are more preferable.

[0016] In the method of the present invention, when the analyte is aglycated protein, it is preferable that a glycation site thereof isdegraded by oxidation with FAOD so that hydrogen peroxide is formed.Also, when the analyte is a glycated peptide or a glycated amino acid,it is preferable that the glycated peptide or the glycated amino acidsimilarly is subjected to the action of FAOD. Moreover, it is preferablethat glycated proteins and glycated peptides are treated with a proteaseprior to the FAOD treatment as necessary.

BRIEF DESCRIPTION OF DRAWINGS

[0017]FIG. 1 is a graph showing the correlation between a molar ratio atwhich a tetrazolium compound and sodium azide are added, a Hbconcentration, and an absorbance in one example of a method formeasurement according the present invention.

[0018]FIG. 2 is a graph showing the relationship between an aging periodand an absorbance of a solution containing a tetrazolium compound andsodium azide in another example of a method for measurement accordingthe present invention.

[0019]FIG. 3 is a graph showing the relationship between an aging periodand an absorbance of a solution containing a tetrazolium compound andsodium azide in still another example of a method for measurementaccording the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0020] The tetrazolium compound used in the present invention preferablycontains ring substituents at least at two positions on its tetrazolering, more preferably at three positions on its tetrazole ring, forexample.

[0021] In the case where the tetrazolium compound contains ringsubstituents at least at two positions on its tetrazole ring asdescribed above, it is preferable that the ring substituents are at the2-position and 3-position on the tetrazole ring. Further, in the casewhere the tetrazolium compound contains ring substituents at threepositions on its tetrazole ring, it is preferable that the ringsubstituents are at the 2-position, 3-position, and 5-position on thetetrazole ring.

[0022] Further, it is preferable that at least two ring substituents ofthe tetrazolium compound have a benzene ring structure. Other than thebenzene ring structure, the ring substituents may have a resonancestructure with S or O being contained in the ring skeleton, for example.Examples of the ring substituents with such a resonance structureinclude a thienyl group, thiazoyl group, and the like.

[0023] Furthermore, it is preferable that the tetrazolium compoundcontains ring substituents at least at three positions on its tetrazolering and at least two of the ring substituents have a benzene ringstructure.

[0024] Still further, it is preferable that at least one ringsubstituent contains a functional group, and a larger number offunctional groups are more preferable.

[0025] As the functional group, an electron-withdrawing functional grouppreferably is used. For example, a halogen group, ether group, estergroup, carboxy group, acyl group, nitroso group, nitro group, hydroxygroup, sulfo group, and the like can be used. Other than these,characteristic groups containing oxygen such as a hydroperoxy group, oxygroup, epoxy group, epidioxy group, oxo group, and the like; andcharacteristic groups containing sulfur such as a mercapto group,alkylthio group, methylthiomethyl group, thioxo group, sulfino group,benzenesulfonyl group, phenylsulfonyl group, p-toluenesulfonyl group,p-tolylsulfonyl group, tosyl group, sulfamoyl group, isothiocyanategroup, and the like also can be used, for example. Among theseelectron-withdrawing functional groups, a nitro group, sulfo group,halogen group, carboxy group, hydroxy group, methoxy group, ethoxy groupare preferable. Further, in addition to the above-describedelectron-withdrawing functional groups, unsaturated hydrocarbon groupssuch as a phenyl group (C₆H₅—), styryl group (C₆H₅CH═CH—), and the likealso can be used, for example. It is to be noted that the functionalgroups may have been ionized by dissociation.

[0026] Still further, it is preferable that the tetrazolium compoundcontains benzene rings at the 2-position and 3-position on its tetrazolering and at least one of the benzene rings contains at least onefunctional group selected from the group consisting of a halogen group,carboxy group, nitro group, hydroxy group, sulfo group, methoxy group,and ethoxy group. It is to be noted here that both the benzene rings mayhave such a functional group. Further, the functional group may becontained at any positions (ortho-, meta-, pra-) on each of the benzenerings. Furthermore, the number of the functional groups is notparticularly limited, and the benzene ring may have either the same ordifferent functional groups.

[0027] Examples of the tetrazolium compound containing ring substituentshaving a benzene ring structure at the 2-position, 3-position, and5-position on its tetrazole ring include:

[0028]2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt;

[0029]2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt;

[0030]2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt;

[0031] 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium salt;

[0032] 3,3′-(1,1′-biphenyl-4,4′-diyl)-bis(2,5-diphenyl)-2H-tetrazoliumsalt;

[0033]3,3′-[3,3′-dimethoxy-(1,1′-biphenyl)-4,4′-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazoliumsalt];

[0034] 2,3-diphenyl-5-(4-chlorophenyl) tetrazolium salt;

[0035] 2,5-diphenyl-3-(p-diphenyl) tetrazolium salt;

[0036] 2,3-diphenyl-5-(p-diphenyl) tetrazolium salt;

[0037] 2,5-diphenyl-3-(4-styrylphenyl) tetrazolium salt;

[0038] 2,5-diphenyl-3-(m-tolyl) tetrazolium salt; and

[0039] 2,5-diphenyl-3-(p-tolyl) tetrazolium salt.

[0040] The tetrazolium compound is not limited to those described above.In addition to the above-described tetrazolium compounds, a tetrazoliumcompound containing ring substituents having a benzene ring structure attwo positions and a ring substituent having a structure other than thebenzene ring structure at one position on its tetrazole ring also may beused. Examples of such a tetrazolium compound include:

[0041] 2,3-diphenyl-5-(2-thienyl) tetrazolium salt;

[0042] 2-benzothiazoyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl) phenyl]-2H-tetrazolium salt;

[0043] 2,2′-dibenzothiazoyl-5,5′-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3′-(3,3′-dimethoxy-4,4′-biphenylene)ditetrazoliumsalt; and

[0044] 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium salt.

[0045] Further, a tetrazolium compound containing ring substituentshaving a benzene ring structure at two positions and a substituent nothaving a ring structure at one position on its tetrazole ring also canbe used. Examples of such a tetrazolium compound include:

[0046] 2,3-diphenyl-5-cyano tetrazolium salt;

[0047] 2,3-diphenyl-5-carboxy tetrazolium salt;

[0048] 2,3-diphenyl-5-methyltetrazolium salt; and

[0049] 2,3-diphenyl-5-ethyl tetrazolium salt.

[0050] Among the above-described tetrazolium compounds, the tetrazoliumcompounds containing three ring substituents are preferable as describedabove. Among these, the tetrazolium compounds containing three ringsubstituents having a benzene ring structure and a large number ofelectron-withdrawing functional groups is more preferable, and2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt is most preferable. It is to be noted here that the above-describedtetrazolium compounds may be a salt or may have been ionized, forexample.

[0051] As the FAOD, FAOD catalyzing a reaction represented by Formula(1) below preferably is used.

R¹—CO—CH₂—NH—R²+H₂O+O₂

→R¹—CO—CHO+NH₂—R²+H₂O₂   (1)

[0052] In Formula (1), R¹ denotes a hydroxyl group or a residue derivedfrom the sugar before glycation (i.e., sugar residue). The sugar residue(R¹) is an aldose residue when the sugar before glycation is aldose, andis a ketose residue when the sugar before glycation is ketose. Forexample, when the sugar before glycation is glucose, it takes a fructosestructure after glycation by an Amadori rearrangement. In this case, thesugar residue (R¹) becomes a glucose residue (an aldose residue). Thissugar residue (R¹) can be represented, for example, by

—[CH(OH)]_(n)—CH₂OH

[0053] where n is an integer of 0 to 6.

[0054] In Formula (1), R² is not particularly limited. However, when thesubstrate is a glycated amino acid, a glycated peptide, or a glycatedprotein, for example, there is a difference between the case where anα-amino group is glycated and the case where an amino group other thanthe α-amino group is glycated.

[0055] In Formula (1), when an α-amino group is glycated, R² is an aminoacid residue or a peptide residue represented by Formula (2) below.

—CHR³—CO—R⁴   (2)

[0056] In Formula (2), R³ denotes an amino-acid side chain group. R⁴denotes a hydroxyl group, an amino acid residue, or a peptide residue,and can be represented, for example, by Formula (3) below. In Formula(3), n is an integer of 0 or more, and R³ denotes an amino-acid sidechain group as in the above.

—(NH—CHR³—CO)_(n)—OH  (3)

[0057] In Formula (1), when an amino group other than the α-amino groupis glycated (i.e., an amino-acid side chain group is glycated), R² canbe represented by Formula (4) below.

—R⁵—CH(NH—R⁶)—CO—R⁷  (4)

[0058] In Formula (4), R⁵ denotes a portion other than the glycatedamino group in the amino-acid side chain group. For example, when theglycated amino acid is lysine, R⁵ is as follows.

—CH₂—CH₂—CH₂—CH₂—

[0059] For another example, when the glycated amino acid is arginine, R⁵is as follows.

—CH₂—CH₂—CH₂—NH—CH(NH₂)—

[0060] In Formula (4), R⁶ denotes hydrogen, an amino acid residue, or apeptide residue, and can be represented, for example, by Formula (5)below. In Formula (5), n denotes an integer of 0 or more, and R³ denotesan amino-acid side chain group as in the above.

—(CO—CHR³—NH)_(n)—H  (5)

[0061] In Formula (4), R⁷ denotes a hydroxyl group, an amino acidresidue, or a peptide residue, and can be represented, for example, byFormula (6) below. In Formula (6), n is an integer of 0 or more, and R³denotes an amino-acid side chain group as in the above.

—(NH—CHR³—CO)_(n)—OH  (6)

[0062] Examples of the FAOD include those produced by the followinggenera, for example: the genus fusarium, the genus Gibberella, the genusPenicillium, the genus Armillaria, the genus Caldariomyces, the genusGanoderma, and the genus Aspergillus. Specific examples include Fusariumoxysporum S-1F4 (FERM BP-5010), Fusarium oxysporum f. sp. lini(IFO NO.5880), Fusarium oxysporum f. sp. batatas (IFO NO. 4468), Fusariumoxysporum f. sp. niveum (IFO NO. 4471), Fusarium oxysporum f. sp.cucumerium (IFO NO. 6384), Fusarium oxysporum f. sp. melongenae (IFO NO.7706), Fusarium oxysporum f. sp. apii (IFO NO. 9964), Fusarium oxysporumf. sp. pini (IFO NO. 9971), Fusarium oxysporum f. sp. fragariae (IFO NO.31180), Gibberella fujikuroi (IFO NO. 6356, 6605), Penicilliumjanthinellum S-3413 (FERM BP-5475), Penicillium janthinellum (IFO NO.4651, 6581, 7905), Penicillium oxalicum (IFO NO. 5748), Penicilliumjavanicum (IFO NO. 4639), Penicillium chrysogenum (IFO NO. 4897),Penicillum cyaneum (IFO NO. 5337), Aspergillus terreus (IFO NO. 6365),Aspergillus terreus GP- 1 (FERM BP-5684), Aspergillus oryzae (IFO NO.4242), and Aspergillus oryzae (IFO NO. 5710).

[0063] Furthermore, examples of commercially available FAOD include aproduct named Fructosyl-Amino Acid Oxidase (FAOX-E) (KikkomanCorporation) and a product named Fructosyl Amine Oxidase (Asahi ChemicalIndustry Co., Ltd.), which specifically act on a glycated amino acidhaving a glycated α-amino group.

[0064] Hereinafter, the method of the present invention will bedescribed in detail with reference to the following examples, in which aglycated protein in blood cells is measured.

[0065] First, whole blood itself is hemolyzed, or a blood cell fractionis separated from whole blood in the usual way such as centrifugationand then hemolyzed, so as to prepare a hemolyzed sample. The method ofcausing the hemolysis is not particularly limited, and can be, forexample, a method using a surfactant, a method using ultrasonic waves, amethod utilizing a difference in osmotic pressure, and a method using afreeze-thawing technique. Among these, the method using a surfactant ispreferable because of its simplicity in operation, etc.

[0066] As the surfactant, for example, non-ionic surfactants such aspolyoxyethylene-p-t-octylphenyl ether (e.g. Triton series surfactants),polyoxyethylene sorbitan alkyl ester (e.g. Tween series surfactants),polyoxyethylene alkyl ether (e.g. Brij series surfactants), and the likecan be used. Specific examples are Triton X-100, Tween-20, Brij 35, andthe like. The conditions of the treatment with the surfactant usuallyare as follows: when the concentration of blood cells in the solution tobe treated is in the range from 1 to 10 vol %, the surfactant is addedso that its concentration in the solution falls in the range from 0.01to 5 wt %, and stirred at room temperature for about several seconds(about 5 seconds) to 10 minutes.

[0067] Next, a tetrazolium compound and sodium azide are added to thehemolyzed sample.

[0068] By adding the tetrazolium compound and sodium azide, thesensitivity becomes about 1.2 to 3 times greater than in the case wherethey are not added.

[0069] When the concentration of blood cells in the solution to betreated is in the range from 0.2 to 2 vol %, the tetrazolium compoundpreferably is added so that its concentration in the solution falls inthe range from 0.005 to 400 mmol/l, more preferably from 0.02 to 100mmol/l, and particularly preferably from 0.1 to 50 mmol/l. Specifically,when the tetrazolium compound is2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt, it preferably is added so that its concentration falls in therange from 0.004 to 16 mmol/l, more preferably from 0.02 to 10 mmol/l,and particularly preferably from 0.1 to 5 mmol/l. Moreover, thetetrazolium compound may be used either alone or in combinations of twoor more types.

[0070] Furthermore, the tetrazolium compound (A) and the sodium azide(B) are added so that they are present at a ratio (molar ratio A: B),for example, in the range from 20:3 to 20:12, preferably 20:5 to 20:11,and more preferably 20:6 to 20:10.

[0071] The tetrazolium compound and sodium azide may be added to thehemolyzed sample simply as they are. However, in terms of simplicity inoperation etc., it is preferable to use a tetrazolium compound solutionobtained by dissolving the tetrazolium compound in a solvent and asodium azide solution obtained by dissolving the sodium azide in asolvent, or a liquid mixture containing both the tetrazolium compoundand sodium azide (i.e., a tetrazolium compound-sodium azide liquidmixture).

[0072] The concentration of the tetrazolium compound (C) or the sodiumazide (D) in the above-described respective solutions can be determinedas appropriate depending on the diluting factor of the solutions whenthey are added to the hemolyzed sample, etc., but the concentration ofthe tetrazolium compound (C) is, for example, in the range from 0.6 to10 mmol/l, preferably from 0.75 to 3 mmol/l, and more preferably from 1to 2.4 mmol/l. When the liquid mixture is to be used, the liquid mixturecontains the tetrazolium compound (C) and the sodium azide (D), forexample, at a ratio (molar ratio C:D) in a range from 20:5 to 20:11,preferably 20:6 to 20:8.

[0073] As the solvent of the above-described solutions, Good's bufferssuch as MOPS, MES, MOPSO, DIPSO, TES, POPSO, and HEPES, a phosphatebuffer, and the like can be used, for example. Among these, MES and MOPSare preferable. The pH of the solvent is, for example, in the range from5.0 to 7.0, preferably 5.5 to 6.5, and more preferably 5.5. Theconcentration of the buffer is, for example, in the range from 1 to 100mmol/l, preferably 1 to 10 mmol/l. The final concentration of the bufferafter being added to the hemolyzed sample is, for example, in the rangefrom 0.7 to 9 mmol/l, preferably from 0.8 to 4.5 mmol/l.

[0074] Moreover, the tetrazolium compound-sodium azide liquid mixtureprepared preferably is left for a certain period before being added tothe hemolyzed sample so as to be aged because this allows still furtherimprovement in sensitivity. According to this aging treatment, thesensitivity becomes, for example, about 1.2 to 3 times greater than inthe case where the aging treatment is not performed.

[0075] In the aging treatment, the treatment temperature preferably isin the range from 40° C. to 60° C., more preferably 50° C. to 60°, andthe treatment period is, for example, at least 6 hours, preferably 6 to120 hours, and more preferably 6 to 72 hours.

[0076] After the tetrazolium compound and sodium azide are added to thehemolyzed sample simply as they are or as the above-described solution,the pretreatment of the hemolyzed sample is carried out, usually byincubating the sample at 40° C. to 60° C. for 6 to 72 hours. Bypretreating the sample with the tetrazolium compound, the influence ofreducing substances and the like contained in the sample on a redoxreaction can be eliminated, whereby the accuracy of measurement isimproved. Although the tetrazolium compound contributes to theimprovement in the accuracy of measurement as described above, it isnecessary that sodium azide coexists with the tetrazolium compound inorder to achieve the improvement in measurement sensitivity as an objectof the present invention. By using the tetrazolium compound and sodiumazide in combination, an effect peculiar to the present invention can beobtained.

[0077] Next, the pretreated hemolyzed sample containing the tetrazoliumcompound and sodium azide is treated with a protease. This proteasetreatment is carried out so that FAOD to be used later can act on theanalyte more easily.

[0078] The type of the protease is not particularly limited, and forexample, serine proteases, thiol proteases, metalloproteinases, and thelike can be used. Specifically, trypsin, proteinase K, chymotrypsin,papain, bromelain, subtilisin, elastase, aminopeptidase, and the likeare preferable. In the case where the glycated protein to be degraded isa glycated hemoglobin, the protease is the one that degrades theglycated hemoglobin selectively, and bromelain, papain, trypsin derivedfrom porcine pancreas, metalloproteinases, and protease derived fromBacillus subtilis, and the like are preferable. Examples of the proteasederived from Bacillus subtilis include a product named Protease N (e.g.,Fluka Chemie AG), a product named Protease N “AMANO” (Amano EnzymeInc.), and the like. Examples of the metalloproteinases includemetalloproteinase (EC 3. 4. 24. 4) derived from the genus Bacillus(e.g., a product named Toyoteam manufactured by Toyobo Co., Ltd.) andthe like. Among these, metalloproteinases, bromelain, and papain aremore preferable, and metalloproteinases are particularly preferable.Thus, a degradation product of a specific protein can be preparedselectively by using a protease that degrades the protein selectively.The protease treatment usually is carried out in a buffer, and theconditions of the treatment are determined as appropriate depending onthe type of the protease used, the type and the concentration of theglycated protein as an analyte, etc.

[0079] As the buffer, CHES, CAPSO, CAPS, phosphate, Tris, EPPS, HEPESbuffers, and the like can be used, for example. The pH of the buffer is,for example, in the range from 6 to 13, preferably from 7 to 11.Moreover, the final concentration of the buffer in the solutionsubjected to the protease treatment is, for example, in the range from1.0 to 10 mmol/l.

[0080] Specifically, when the pretreated hemolyzed sample is treatedusing a metalloproteinase as the protease, the protease treatmentusually is carried out under the conditions as follows: theconcentration of the metalloproteinase in the reaction solution in therange from 0.1 to 40 MU/l; the concentration of blood cells in thereaction solution in the range from 0.05 to 15 vol %; the reactiontemperature in the range from 15° C. to 37° C.; the reaction period inthe range from 1 minute to 24 hours; and the pH in the range from 6 to12.

[0081] Furthermore, when the pretreated hemolyzed sample is treatedusing protease K as the protease, the protease treatment usually iscarried out under the conditions as follows: the concentration of theprotease in the reaction solution in the range from 10 to 300 KU/l; theconcentration of blood cells in the reaction solution in the range from0.05 to 15 vol %; the reaction temperature in the range from 15° C. to37° C.; the reaction period in the range from 1 minute to 24 hours; andthe pH in the range from 6 to 12. Moreover, the type of the buffer isnot particularly limited, and for example, Tris-HCl buffer, EPPS buffer,PIPES buffer, and the like can be used.

[0082] Next, the degradation product obtained by the protease treatmentis treated with the FAOD. The reaction shown by Formula (1) above iscatalyzed by this FAOD treatment.

[0083] Similarly to the above-described protease treatment, this FAODtreatment preferably is carried out in a buffer. The conditions of theFAOD treatment are determined as appropriate depending on the type ofthe FAOD used, the type and the concentration of the glycated protein asan analyte, and the like.

[0084] Specifically, the FAOD treatment is carried out, for example,under the following conditions: the concentration of the FAOD in thereaction solution in the range from 50 to 50,000 U/l, the concentrationof the blood cells in the reaction solution in the range from 0.01 to 1vol %, the reaction temperature in the range from 15° C. to 37° C., thereaction period in the range from 1 to 60 minutes, and the pH in therange from 6 to 9. Moreover, the type of the buffer is not particularlylimited, and the same buffers as in the protease treatment also can beused in the FAOD treatment.

[0085] Next, the hydrogen peroxide formed by the FAOD treatment ismeasured by a redox reaction using POD and the color-developingsubstrate.

[0086] As the color-developing substrate,N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt, orthophenylenediamine (OPD), a substrate in which aTrinder's reagent and 4-aminoantipyrine are combined, and the like canbe used, for example. Examples of the Trinder's reagent include phenols,phenol derivatives, aniline derivatives, naphthols, naphtholderivatives, naphthylamine, and naphthylamine derivatives. Furthermore,in place of the aminoantipyrine, it is possible to use aminoantipyrinederivatives, vanillin diamine sulfonic acid, methylbenzothiazolinonehydrazone (MBTH), sulfonated methylbenzothiazolinone hydrazone (SMBTH),and the like. Among these color-developing substrates,N-(carboxymethylaminocarbonyl)-4,4′-bis (dimethylamino)diphenylaminesodium salt is particularly preferable.

[0087] The redox reaction usually is carried out in a buffer. Theconditions of the reaction are determined as appropriated depending onthe concentration of the hydrogen peroxide formed, etc. The conditionsare usually as follows: the concentration of the POD in the reactionsolution in the range from 10 to 100,000 IU/l; the concentration of thecolor-developing substrate in the range from 0.005 to 30 mmol/l; thereaction temperature in the range from 15° C. to 37° C.; the reactionperiod in the range from 0.1 to 30 minutes; and the pH in the range from5 to 9. Moreover, the type of the buffer is not particularly limited,and for example, the same buffers as in the protease treatment and theFAOD treatment can be used.

[0088] In the redox reaction, for example, when the color-developingsubstrate is used, the amount of the hydrogen peroxide can be determinedby measuring the degree of the color developed (i.e. absorbance) in thereaction solution with a spectrophotometer. Then, for example, theamount of the glycated protein in the sample can be determined using theconcentration of the hydrogen peroxide and a calibration curve or thelike.

[0089] The degree of the color developed can be determined not only bymeasuring the absorbance but also by optical measurement such asmeasurement of reflectance or the like. Moreover, the amount of thehydrogen peroxide can be determined not only by the above-describedenzymatic method using the POD etc. but also by an electrical method,for example.

[0090] Thus, by adding a tetrazolium compound and sodium azide, themeasurement of a glycated protein using a redox reaction can be carriedout with high sensitivity.

[0091] The order of adding a tetrazolium compound and sodium azide isnot particularly limited. However, since aging them enhances the effectof the present invention, they preferably are mixed with each other inadvance.

[0092] Furthermore, as described above, the analyte is not particularlylimited as long as a redox reaction is utilized. Examples of the analyteother than the above-described glycated proteins include glycatedpeptides, glycated amino acids, glucose, cholesterol, uric acid,creatinine, sarcosine, and glycerol. When the amount of each of theabove-described examples of the analyte is measured, measurement can becarried out, for example, by adding a tetrazolium compound and sodiumazide to a measurement sample in the same manner as described above,then forming a reducing substance or an oxidizing substance derived fromthe analyte in the following manner, and measuring the amount of thereducing substance or oxidizing substance using a redox reaction.

[0093] When the measurement is carried out by forming hydrogen peroxide,the hydrogen peroxide may be formed, for example, by action of a glucoseoxidase on the glucose; a cholesterol oxidase on the cholesterol; auricase on the uric acid; a sarcosine oxidase on the creatinine; asarcosine oxidase on the sarcosine; or a glycerol oxidase on theglycerol; respectively. The amount of the hydrogen peroxide can bemeasured in the same manner as above. Moreover, glycated peptides andglycated amino acids can be measured, for example, in the same manner asin the measurement of the glycated proteins.

[0094] Furthermore, when the amount of the analyte is determined byforming a reducing substance derived from the analyte, measuring theamount of the reducing substance by a redox reaction, and thendetermining the amount of the analyte from the amount of the reducingsubstance, the measurement can be carried out, for example, in thefollowing manner.

[0095] When the analyte is glucose, for example, a reducing substancesuch as NADH or NADPH is formed using glucose dehydrogenase in thepresence of NAD, NADP, or the like. Then, the NADH or NADPH as areducing substance derived from the analyte is measured by a redoxreaction, using, for example, diaphorase and a substrate that developscolor by reduction. Then, as described above, the amount of the analytein the sample can be determined, for example, using the concentration ofthe reducing substance derived from the analyte and a calibration curveor the like. Furthermore, for example, cholesterol dehydrogenase can beused when the analyte is cholesterol, and sarcosine dehydrogenase can beused when the analyte is sarcosine.

[0096] As the substrate that develops color by reduction, although notparticularly limited, for example, 2,6-dichlorophenolindophenol and thelike can be used. Moreover, in order to obtain measured values with moreexcellent reliability, it is preferable to measure an absorbance beforemeasuring the reducing substance derived from the analyte, for example.

EXAMPLES Example 1 and Comparative Example 1

[0097] First, blood having a Hb concentration of 150 g/l (HbA1c 5.8%)was hemolyzed by adding 0.3 ml of the following hemolysis reagent toprepare a hemolyzed sample. Then, 65 μl of the following first reagentscontaining 3g/l sodium azide aqueous solution at predeterminedconcentrations (0 mmol/l, 0.30 mmol/l, 0.616 mmol/l, 0.924 mmol/l, 1.232mmol/l, and 1.54 mmol/l) were added to mixtures of 25 μl of thehemolyzed sample and 20 μl of purified water, respectively. Theresultant mixtures were incubated at 37° C. for 5 minutes. The firstreagents were aged at 50° C. for 20 hours before being added to thehemolyzed sample. Subsequently, 45 μl of the following color-developingreagent A further was added, and the resultant mixtures were incubatedat 37° C. for 3 minutes. Thereafter, with regard to the thus-obtainedreaction solutions, the absorption was measured at the main wavelengthof 751 nm and the sub-wavelength of 805 nm using a biochemical automaticanalysis apparatus (product name Bio Majesty, manufactured by JapanElectron Optics Laboratory Co. Ltd.). The results are shown in Table 1.The mixture containing no sodium azide (0 g/l) was regarded asComparative Example 1. (Hemolysis Reagent: pH 9.0) CHES buffer 380mmol/l Polyoxyethylene lauryl ether 24 g/l (First Reagent) 100 mmol/lMOPS (pH 6.0) 0.15 ml 13 MU/l metalloproteinase 0.60 ml 10 mmol/ltetrazolium compound 0.60 ml 20 mmol/l CaCl₂ 0.045 ml  3 g/l NaN₃predetermined amount distlled water remaining portion total amount  3.0ml

[0098] As the metalloproteinase, metalloproteinase derived from thegenus Bacillus was used. As the tetrazolium compound,2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt (product name WST-3, manufactured by Dojindo Laboratories,hereinafter referred to as “WST-3”) was used. (Color-Developing ReagentA) FAOD (product name Fructosyl Amino Acid Oxidase, 26.0 KU/l ARKRAY,INC., hereinafter the same) POD (Toyobo Co., Ltd., hereinafter the same)77.6 KU/l Color-developing substrate (product name DA-64, 0.052 mmol/lWako Pure Chemical Industries, Ltd., hereinafter the same) Tris-HClbuffer (pH 6.9) 200 mmol/l

[0099] TABLE 1 Final concentration Concentration of of sodium azidetetrazolium compound in first reagent in first reagent (mmol/l) (mmol/l)Absorbance Com. Ex. 1 0 2 0.0124 Ex. 1 0.300 2 0.0476 0.616 2 0.05180.924 2 0.0475 1.232 2 0.0418 1.540 2 0.0275

[0100] As can be seen from Table 1, in Example 1, by conducting themeasurement in the presence of the tetrazolium compound and sodiumazide, the absorbance higher than that in Comparative Example 1 wasobtained because the amount of the color developed by thecolor-developing substrate DA-64 increased. Thus, it can be said thatthe method according to the present invention can improve themeasurement sensitivity.

Example 2 and Comparative Example 2

[0101] In Example 2, WST-3 and sodium azide were added so that they werepresent at various ratios, and further improvement in measurementsensitivity by aging them was confirmed.

[0102] (Reagent Sample)

[0103] Reagent samples (A) to (E) were prepared by mixing WST-3 andsodium azide so that they were present at the following ratios. Then,the samples (B) to (E) were incubated at 40° C. for 20 hours.Subsequently, the samples (A) to (E) were maintained at the temperaturenot higher than 4° C. and metalloproteinase was added to the samples sothat its concentration became 2.66 MU/l.

[0104] (Ratio Between WST-3 and Sodium Azide)

Example 2

[0105] (A) (B) (C) (D) (E) WST-3 (mmol/l) 2.0 2.0 2.0 2.7 1.3 NaN₃(mmol/l) 0.7 0.7 1.4 1.4 0.7 WST-3:NaN₃ (molar ratio) 3:1 3:1 1.43:11.9:1 1.9:1 MOPS pH 6.5 (mmol/l) 5.5 5.5 5.5 5.5 5.5 CaCl₂ (mmol/l) 5.55.5 5.5 5.5 5.5 NaCl (mmol/l) 0.33 0.33 0.33 0.33 0.33 Aging — ∘ ∘ ∘ ∘

[0106] (Hemolyzed Solution)

[0107] Distilled water was added to blood cells. The mixture was frozenand then thawed to hemolyze the blood cells In the hemolyzed solutionthus obtained, the Hb concentration was 100 g/l and HbA1c was 4.9%.

[0108] The hemolyzed solution thus prepared was pretreated by adding thefollowing pretreatment reagent so as to have the following compositions.Hb concentration 6.7 g/l 13.4 g/l 20.1 g/l 33.5 g/l Hemolyzed solution 50 μl 100 μl 150 μl 250 μl Pretreatment reagent 434 μl 434 μl 434 μl434 μl Distilled water 266 μl 216 μl 166 μl  66 μl total amount 750 μl

[0109] The pretreatment reagent was prepared by adding a surfactant to abuffer so that its concentration became 20 wt %. As the surfactant,polyoxyethylene lauryl ether (product name Nikkol, manufactured by NihonSurfactant Kogyo K.K.) was used. The buffer used was a mixed buffer (pH9.5) obtained by mixing a glycinamide buffer and a glycine solution sothat the final concentration of glycinamide became 200 mM and the finalconcentration of glycine became 40 mM. (Color-Developing Reagent B) FAOD25.9 KU/l POD 77.6 KU/l Color-developing substrate 0.0518 mmol/lTris-HCl buffer (pH 6.9) 200 mmol/l

[0110] (Measuring procedure)

[0111] First, the pretreated hemolyzed solutions were diluted 2.4-foldwith distilled water, and 20 μL of these diluted solutions were mixedwith 60 μl of each of the reagent samples and 50 μl of thecolor-developing reagent B. The resultant mixtures were allowed to reactat 37° C. for 15 minutes. Thereafter, the absorption was measured at themain wavelength of 751 nm and the sub-wavelength of 805 nm using abiochemical automatic analysis apparatus (product name JCA-BM 8,manufactured by Japan Electron Optics Laboratory Co. Ltd.). The resultsare shown in Table 2 below and in the graph shown in FIG. 1. The graphof FIG. 1 shows the relationship between a molar ratio between thetetrazolium compound and sodium azide, a Hb concentration, and anabsorbance. TABLE 2 Example 2 Hb concentration (A) (B) (C) (D) (E)   0g/l 0.1542 0.1628 0.1469 0.1403 0.1647  6.7 g/l 0.0034 0.0101 0.00870.0099 0.0097 13.4 g/l 0.0063 0.0180 0.0146 0.0173 0.0171 20.1 g/l0.0094 0.0259 0.0202 0.0249 0.0243 33.5 g/l 0.0127 0.0384 0.0278 0.03740.0349

[0112] As shown in Table 2 and FIG. 1, high absorbance was exhibited byadding the tetrazolium compound and sodium azide. Moreover, furtherimprovement in absorbance was achieved by conducting incubation (aging)((B) to (E)). Moreover, since the absorbance increased in keeping withthe Hb concentration, it can be said that the absorbance did notincrease due to the absorption of the tetrazolium compound and sodiumazide themselves but increased because the absorbance of thecolor-developing substrate DA-64 increased by adding the tetrazoliumcompound and sodium azide. Furthermore, the significant increase inabsorbance was observed when the tetrazolium compound and sodium azidewere added so that they were present at the molar ratio of 3:1 and theywere aged as well. Thus, because the amount of color developed by thecolor-developing substrate increased by adding the tetrazolium compoundand sodium azide and it increased still further by conducting aging, itcan be said that the measurement sensitivity was improved.

Example 3

[0113] In Example 3, a tetrazolium compound and sodium azide were agedin a solution to examine how this affects the improvement in measurementsensitivity.

[0114] First, a liquid mixture containing 3.33 mmol/l of WST-3 and0.0833 g/l of sodium azide was prepared and then aged by being incubatedat 60° C. Then, samples were taken predetermined periods (0 hour, 1hour, 6 hours, 14 hours, 16 hours, and 18 hours) after the start of theincubation. These samples were used as reagent samples.

[0115] On the other hand, a hemolyzed solution (Hb concentration: 100g/l, HbA1c: 4.9%) was prepared in the same manner as in Example 2. Then,predetermined amounts (2 μl, 5 μl, 10 μl, 20 μl, and 30 μl) of hemolyzedsolution were mixed with 300 μl of the following pretreatment reagent toprepare substrate Hb solutions (A to E).

[0116] (Pretreatment Reagent)

[0117] Liquid mixture (pH 9.4) of 80 mmol/l CHES and 30 mmol/l MOPS 9g/l of polyoxyethylene lauryl ether

[0118] First, the respective substrate Hb solutions were diluted 2-foldwith distilled water, and 20 μL of these diluted solutions were mixedwith 65 μl of each of the following second reagents respectivelycontaining the above-described reagent samples and 45 μl of thecolor-developing reagent C. The resultant mixtures were allowed to reactat 37° C. for 15 minutes. Thereafter, the absorption at the mainwavelength of 751 nm and the sub-wavelength of 805 nm was measured usingthe above-described biochemical automatic analysis apparatus. Theresults are shown in Table 3 below and FIG. 2. FIG. 2 is a graph showingthe relationship between an aging period and an absorbance. (SecondReagent) 1 mol/l CaCl₂ 0.035 ml 5 mol/l NaCl 0.280 ml 30 mmol/l MES (pH5.5)  1.40 ml 100 MU/l metalloproteinase  0.7 ml distlled water 0.385 mltotal amount  7.0 ml (Color-Developing Reagent C) FAOD 25.9 KU/l POD77.6 KU/l Color-developing substrate 0.0518 mmol/l Tris-HCl buffer (pH7.0) 300 mmol/l

[0119] TABLE 3 Final concentration of Hb Aging period (A) (B) (C) (D)(E) (hour) 0.10 g/l 0.25 g/l 0.50 g/l 0.96 g/l 1.40 g/l  0 0.0003 0.00070.0015 0.0037 0.0065  1 0.0003 0.0008 0.0017 0.0042 0.0073  6 0.00080.0020 0.0039 0.0084 0.0129 14 0.0009 0.0020 0.0039 0.0084 0.0130 160.0008 0.0020 0.0039 0.0083 0.0128 18 0.0008 0.0020 0.0039 0.0083 0.0129

[0120] As shown in Table 3 and FIG. 2, it was found that furtherimprovement in measurement sensitivity can be achieved by using thesolution containing the tetrazolium compound and sodium azide afteraging it, regardless of the Hb concentration. Furthermore, thesensitivity reached its maximum when the aging period was 6 hours orlonger.

Example 4

[0121] In Example 4, a tetrazolium compound and sodium azide were agedin a solution to examine how this affects the improvement in measurementsensitivity.

[0122] First, the following liquid mixture containing WST-3 and sodiumazide was prepared and then aged by being incubated at predeterminedtemperatures (30° C., 40° C., 50° C., and 60° C.) for predeterminedperiods (0 hour, 6 hours, 15 hours, 24 hours, 48 hours, and 72 hours) toprepare reagent samples. (Composition of Liquid Mixture) WST-3 2.20mmol/l Sodium azide 0.05 g/l MOPS (pH 6.5) 5.50 mmol/l CaCl₂ 5.50 mmol/lNaCl 330 mmol/l

[0123] On the other hand, substrate Hb solutions (Hb concentration: 100g/l) were prepared in the same manner as in Example 2 and diluted 2-fold(by volume) with purified water to prepare diluted solutions. On theother hand, metalloproteinase solutions were prepared by mixing 9 ml ofthe above-described respective reagent samples with 1 ml ofmetalloproteinase (2.7 MU/l). Then, 20 μL of the diluted solutions weremixed with 65 μl of each of the metalloproteinase solutions and 45 μl ofthe color-developing reagent A. The resultant mixtures were allowed toreact at 37° C. for 15 minutes. Thereafter, the absorption was measuredat the main wavelength of 751 nm and the sub-wavelength of 805 nm usingthe above-described biochemical automatic analysis apparatus. Theresults are shown in Table 4 below and FIG. 3. FIG. 3 is a graph showingthe relationship between an aging period and an absorbance. TABLE 4Aging period Aging temperature (hour) 30° C. 40° C. 50° C. 60° C.  00.0078 0.0078 0.0078 0.0078  6 0.0211 0.0244 0.0253 0.0277 24 0.02590.0270 0.0323 0.0320 48 0.0271 0.0286 0.0288 0.0301 72 0.0302 0.03110.0311 0.0308

[0124] As shown in Table 4 and FIG. 3, the absorbance increased rapidlywhen the aging was conducted at 60° C. Therefore, it can be said thatthe measurement sensitivity can be improved with shorter period of agingas the aging temperature becomes higher.

[0125] Industrial Applicability

[0126] As specifically described above, the method for measurement usinga redox reaction according to the present invention is excellent inmeasurement sensitivity because it uses a tetrazolium compound andsodium azide. Therefore, by applying the method of the present inventionto, for example, the measurement of HbA1c contained in erythrocytes, itbecomes possible to realize the measurement with higher accuracy than inconventional methods, which further increases the importance of HbAlc asan index in the diagnosis and the like of diabetes.

1. A method of measuring an analyte in a sample using a redox reaction,comprising: measuring an amount of a reducing substance or an oxidizingsubstance derived from the analyte in the presence of a tetrazoliumcompound and sodium azide using the redox reaction; and determining anamount of the analyte from the amount of the reducing substance oroxidizing substance thus measured.
 2. The method according to claim 1,wherein the tetrazolium compound (A) and the sodium azide (B) arepresent at a ratio (molar ratio A:B) in a range from 20:3 to 20:12. 3.The method according to claim 1, wherein a final concentration of thetetrazolium compound in a reaction solution of the redox reaction is ina range from 0.5 to 2.5 mmol/l, and a final concentration of the sodiumazide in the reaction solution is in a range from 0.13 to 1.3 mmol/l. 4.The method according to claim 1, wherein a solution containing thetetrazolium compound and the sodium azide is aged and is then added tothe sample.
 5. The method according to claim 4, wherein the solution isaged at a temperature in a range from 20° C. to 60° C.
 6. The methodaccording to claim 4, wherein the solution is aged for 6 to 120 hours.7. The method according to claim 1, wherein the tetrazolium compound is2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium salt.
 8. The method according toclaim 1, wherein the analyte is at least one selected from the groupconsisting of glycated proteins, glycated peptides, and glycated aminoacids.
 9. The method according to any one of claims 8, wherein theglycated proteins are glycated hemoglobins.
 10. The method according toclaim 1, wherein the oxidizing substance derived from the analyte ishydrogen peroxide.
 11. The method according to claim 10, wherein thehydrogen peroxide is formed by a reaction between a glycation site of aglycated protein and a fructosyl amino acid oxidase.
 12. The methodaccording to claim 10, wherein the amount of the hydrogen peroxide asthe oxidizing substance is measured using an oxidase and a substratethat develops color by oxidation.
 13. The method according to claim 12,wherein the amount of the hydrogen peroxide is measured by opticalmeasurement of a degree of color developed by the substrate thatdevelops color by oxidation.
 14. The method according to claim 13,wherein the optical measurement is measurement of an absorbance or areflectance.